U.S. patent number 3,972,859 [Application Number 05/555,126] was granted by the patent office on 1976-08-03 for novel decapeptide amide analogs of leuteinizing hormone-releasing hormone.
This patent grant is currently assigned to Takeda Chemical Industries, Ltd.. Invention is credited to Masahiko Fujino, Tsunehiko Fukuda, Susumu Shinagawa.
United States Patent |
3,972,859 |
Fujino , et al. |
August 3, 1976 |
Novel decapeptide amide analogs of leuteinizing hormone-releasing
hormone
Abstract
The novel decapeptide amide derivatives of the formula wherein
R.sub.1 is Tyr or Phe; R.sub.2 is D-Nle, D-Nva, D-Abu, D-Phe,
D-Ser, D-Thr or D-Met and R.sub.3 is Leu, Ile or Nle have a strong
ovulation inducing activity.
Inventors: |
Fujino; Masahiko (Takarazuka,
JA), Shinagawa; Susumu (Osaka, JA), Fukuda;
Tsunehiko (Suita, JA) |
Assignee: |
Takeda Chemical Industries,
Ltd. (Osaka, JA)
|
Family
ID: |
12221217 |
Appl.
No.: |
05/555,126 |
Filed: |
March 4, 1975 |
Foreign Application Priority Data
|
|
|
|
|
Mar 8, 1974 [JA] |
|
|
49-27442 |
|
Current U.S.
Class: |
530/313; 514/800;
930/DIG.695; 930/DIG.785; 530/328; 930/DIG.783; 930/20; 930/21;
930/130 |
Current CPC
Class: |
C07K
7/23 (20130101); C07K 5/1005 (20130101); Y10S
930/13 (20130101); Y02P 20/55 (20151101); A61K
38/00 (20130101); Y10S 514/80 (20130101) |
Current International
Class: |
C07K
5/103 (20060101); C07K 7/00 (20060101); C07K
7/23 (20060101); C07K 5/00 (20060101); A61K
38/00 (20060101); C07C 103/52 (); A61K
037/00 () |
Field of
Search: |
;260/112.5LH |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Gotts; Lewis
Assistant Examiner: Suyat; Reginald J.
Attorney, Agent or Firm: Wenderoth, Lind & Ponack
Claims
What is claimed is:
1. A compound of the formula
wherein R.sub.1 is Tyr or Phe; R.sub.2 is D-Nle, D-Nva, D-Abu,
D-Phe, D-Ser, D-Thr or D-Met and R.sub.3 is Leu, Ile or Nle.
2. The compound as claimed in claim 1, wherein R.sub.1 is Tyr,
R.sub.2 is D-Nle and R.sub.3 is Leu.
3. The compound as claimed in claim 1, wherein R.sub.1 is Tyr,
R.sub.2 is D-Ser and R.sub.3 is Leu.
4. The compound as claimed in claim 1, wherein R.sub.1 is Tyr,
R.sub.2 is D-Abu and R.sub.3 is Leu.
5. The compound as claimed in claim 1, wherein R.sub.1 is Tyr,
R.sub.2 is D-Nva and R.sub.3 is Leu.
6. The compound as claimed in claim 1, wherein R.sub.1 is Tyr,
R.sub.2 is D-Thr and R.sub.3 is Leu.
7. The compound as claimed in claim 1, wherein R.sub.1 is Tyr,
R.sub.2 is D-Phe and R.sub.3 is Leu.
8. The compound as claimed in claim 1, wherein R.sub.1 is Tyr,
R.sub.2 is D-Met and R.sub.3 is Leu.
9. The compound as claimed in claim 1, wherein R.sub.1 is Phe,
R.sub.2 is D-Phe and R.sub.3 is Leu.
10. The compound as claimed in claim 1, wherein R.sub.1 is Tyr,
R.sub.2 is D-Abu and R.sub.3 is Ile.
11. The compound as claimed in claim 1, wherein R.sub.1 is Phe,
R.sub.2 is D-Ser, R.sub.3 is Nle.
12. The compound as claimed in claim 1, wherein R.sub.1 is Phe,
R.sub.2 is D-Nle, R.sub.3 is Nle.
13. The compound as claimed in claim 1, wherein R.sub.1 is Phe,
R.sub.2 is D-Nva, R.sub.3 is Ile.
14. The compound as claimed in claim 1, wherein R.sub.1 is Phe,
R.sub.2 is D-Nva, R.sub.3 is Leu.
Description
DECAPEPTIDE AMIDES
The present invention relates to novel decapeptide amide
derivatives having strong ovulation inducing activity, which are
represented by the formula:
wherein R.sub.1 is Tyr or Phe; R.sub.2 is D-Nle, D-Nva, D-Abu,
D-Phe, D-Ser, D-Thr or D-Met and R.sub.3 is Leu, Ile or Nle.
The present invention relates also to a method for producing the
decapeptide amide derivatives (I).
In the present specification and the claims, amino acids and
peptides and their activated carboxyl or protective groups are
designated by abbreviations which are in common usage in the
particular field of art or which have been approved by Committee on
Biochemical Nomenclature of IUPAC-IUB. Amino acid is in the
L-configuration unless otherwise designated.
The following abbreviations are used, for instance.
Abu: .alpha.-Aminobutyric acid
Arg: Arginine
Boc: t-Butoxycarbonyl
Bzl: Benzyl
Dcc: n,n'-dicyclohexylcarbodiimide
Gly: Glycine
His: Histidine
Honb: n-hydroxy-5-norbornene-2,3-dicarboximide
Ile: Isoleucine
Leu: Leucine
Nle: Norleucine
Nva: Norvaline
Met: Methionine
Ome: Methyl ester
Obzl: Benzyl ester
Onb: n-hydroxy-5-norbornene-2,3-dicarboximide ester
Osu: N-Hydroxysuccinimide ester
Phe: Phenylalanine
Pro: Proline
(Pyr)Glu: Pyroglutamic acid
Ser: Serine
Thr: Threonine
Tos: Tosyl
Trp: Tryptophan
Tyr: Tyrosine
It was known for many years that the hypothalamus contains factors
which, at a higher level, control the secretion of tropic hormones
from the pituitary. Subsequent to the isolation of a
thyrotropin-releasing hormone (TRH), luteinizing hormone releasing
hormone (LH-RH) has been extracted in pure form from pigs and sheep
and shown to be a decapeptide of the structure:
(Pyr)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.sub.2. [A. V.
Schally et al., Biochem. Biophys. Res. Commun., 43, 1334(1971): R.
Guillemin et al., Proc. Nat. Acad. Sci., U.S.A., 69, 278(1972)].
This finding has been followed by the synthesis of a number of
similar peptides and biological tests have also been performed on
these analogous peptides. However, even a minor modification in the
above amino acid composition diminishes seriously the physiological
activity of the peptide and the above chemical structure has been
considered to be essential to the genesis of maximal physiological
activity. [A. V. Schally et al., Biochem. Biophys. Res. Commun., 4,
366 (1972)].
Recently Monahan et al published in "Biochemistry", vol. 12, No.
23, pages 4616-4620(1973) that among the LH-RH analogs such as
[Ala.sup.6 ]LH-RH, [D-Ala.sup.6 ]LH-RH, [Val.sup.6 ]LH-RH,
[D-Val.sup.6 ]LG-RH, [Pro.sup.6 ]LH-RH and [D-Pro.sup.6 ]LH-RH
which were synthesized by them, the [D-Ala.sup.6 ]LH-RH exhibited
the strongest activity to be 350-450 % of the potency of the parent
LH-RH. The literature teaches further that LH-RH analogs having at
the 6-position D-amino acid with larger bulk of side chains than
D-Ala are less potent than the parent hormone. In spite of the
state of the art mentioned above, the present inventors synthesized
the polypeptides (I) having at the 6-position D-amino acid with
larger bulk of side chains than D-Ala and tested for their LH-RH
activity and found unexpectedly that the present polypeptides (I)
are at least 1,000 % more potent than parent LH-RH. The present
invention is the culmination of those unexpected findings.
Therefore, it is the main object of the present invention to
provide novel decapeptide amide derivatives (I) which have strong
ovulation inducing activity.
Another object of the present invention is to provide a method for
the production of the decapeptide amide derivatives (I).
Further objects of the present invention will be made clear in
accordance with the description mentioned hereinafter in this
specification.
The decapeptide amide derivative (I) is produced by a method
characterized by that a reagent (A) -- L-Pyroglutamic acid or a
peptide fragment which has an L-pyroglutamic acid unit (i.e.
(Pyr)Glu-) at its N-terminal end and at the same time which, from
thereon, comprises the above amino acid sequence -- is condensed
with a reagent (B) -- an amine component which corresponds to the
balance of the decapeptide amide derivative (I) -- the two reagents
(A) and (B) being optionally protected by a protecting group or
groups, and then the protecting group or groups if any are
removed.
Thus, the reagent (A) is L-pyroglutamic acid or a peptide fragment
which has an L-pyroglutamic acid unit at its N-terminal end and at
the same time which from thereon comprises amino acid sequence of
formula (I), and the reagent (B) to be condensed with the reagent
(A) is an amine component which corresponds to the balance of the
decapeptide amide derivative (I), the reagents (A) and (B) being
optionally protected.
Basic combinations of the reagent (A) and the reagent (B) are
exemplified in the following Table 1.
Table 1 ______________________________________ Reagent (A) (B)
Combina- tion ______________________________________ 1 (Pyr)Glu-OH
H-His-Trp-Ser-R.sub.1 -R.sub.2 - R.sub.3 -Arg-Pro-Gly-NH.sub.2 2
(Pyr)Glu-His-OH H-Trp-Ser-R.sub.1 -R.sub.2 -R.sub.3 -
Arg-Pro-Gly-NH.sub.2 3 (Pyr)Glu-His- H-Ser-R.sub.1 -R.sub.2
-R.sub.3 -Arg- Trp-OH Pro-Gly-NH.sub.2 4 (Pyr)Glu-His- H-R.sub.1
-R.sub.2 -R.sub.3 -Arg-Pro- Trp-Ser-OH Gly-NH.sub.2 5 (Pyr)Glu-His-
H-R.sub.2 -R.sub.3 -Arg-Pro- Trp-Ser-R.sub.1 -OH Gly-NH.sub.2 6
(Pyr)Glu-His- H-R.sub.3 -Arg-Pro-Gly- Trp-Ser-R.sub.1 -R.sub.2 -OH
NH.sub.2 7 (Pyr)Glu-His- H-Arg-Pro-Gly-NH.sub.2 Trp-Ser-R.sub.1
-R.sub.2 - R.sub.3 -OH 8 (Pyr)Glu-His-Trp- H-Pro-Gly-NH.sub.2
Ser-R.sub.1 -R.sub.2 -R.sub.3 -Arg- OH 9 (Pyr)Glu-His-Trp-
H-Gly-NH.sub.2 Ser-R.sub.1 -R.sub.2 -R.sub.3 -Arg- Pro-OH 10
(Pyr)Glu-His-Trp- NH.sub.3 Ser-R.sub.1 -R.sub.2 -R.sub.3 -Arg-
Pro-Gly-OH ______________________________________
It has also been known that a protected L-glutamyl group shown by
the general formula (II):
[wherein R.sub.4 is an alkoxy group (e.g. methoxy, ethoxy,
n-propoxy, i-propoxy, n-butoxy, etc.), an aralkyloxy group (e.q.
benzyloxy, etc.) or amino] is easily converted to the
L-pyroglutamyl group itself: ##STR1## by the contact with a base
(e.g. ammonia, etc.) or an acid (e.g. acetic acid etc.) and that
the group (II) is equivalent to L-pyroglutamyl group itself in this
respect. In the method of the present invention, it is to be
construed that the L-pyroglutamyl (i.e. (Pyr)Glu-) of the reagent
(A) includes not only the L-pyroglutamyl group itself but also the
protected L-glutamyl group of the formula (II). In case when
(Pyr)Glu- of the reagent (A) represents the group (II), the group
(II) is easily converted to L-pyroglutamyl group itself in
accordance with per se known means.
The condensation reaction according to this invention can be
carried out by condensing means known for the formation of peptide
linkages. Among such means of condensation are DCC/HONB process
[Belgian Pat. No. 796,399], the azide process, chloride process,
acid anhydride process, mixed acid anhydride process, DCC process,
active ester process, Woodward reagent K process, carbodiimidazole
process, oxidation-reduction process and others [The Peptides,
Vol.1(1966), Schroder and Lubke, Academic Press, New York,
U.S.A.].
Prior to the condensation reaction, one may protect the carboxyl
and amino groups which should not be involved in the contemplated
reaction or activate the carboxyl or/and amino groups which will
take part in the reaction, by means which are known per se. The
carboxyl groups in the starting material may be protected in the
form of metal salts (e.g. sodium and potassium salts) or esters
(e.g. methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl or t-amyl
esters).
Protective groups for amino groups in the starting materials may be
any of conventional protecting groups of amino groups in peptide
synthesis, e.g. benzyloxycarbonyl, t-butoxycarbonyl,
isobornyloxycarbonyl, etc. The imino group of histidine may be
protected with any of conventional protecting group such as benzyl,
tosyl, 2,4-dinitrophenol, t-butoxycarbonyl or carbobenzoxy. The
hydroxyl group of serine may be protected with a conventional
protective group such as benzyl, t-butyl and other ether-forming
groups. The hydroxyl group of tyrosine may be protected with
benzyl, t-butyl and other ether-forming groups; the guanidino group
of arginine may be protected with such groups as nitro, tosyl,
carbobenzoxy, isobornyloxycarbonyl or adamantyloxycarbonyl. As
examples of activated carboxyl groups in starting materials, there
may be mentioned the corresponding acid anhydride, azide, active
esters [esters with alcohols (e.g. pentachlorophenol,
2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol,
p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide,
N-hydroxysuccinimide, N-hydroxyphthalimide or
N-hydroxybenztriazole)], etc. The activated amino groups in
starting materials may for example be the corresponding phosphoric
acid amide.
The following table shows some exemplary combinations of such forms
of carboxyl and amino groups in materials (A) and (B).
Table 2
__________________________________________________________________________
Starting Material Exemplary (A) (B) combinations COOH NH.sub.2 COOH
NH.sub.2
__________________________________________________________________________
1* Free Protected Protected Free 2 Activated Protected Free Free 3
Free Protected Protected Activated
__________________________________________________________________________
(Note) In the case designated by an asterisk*, a dehydrating agent
(e.g. a carbodiimide reagent such as dicyclohexyl-carbodiimide) is
preferably present in the reaction system. A mode of practice of
this invention may be written as follows. ##STR2## ##STR3##
##STR4## This reaction may be conducted in the presence of a
solvent. The solvent can be selected from those known to be useful
for peptide condensation reactions. Thus, anhydrous or aqueous
dimethylformamide, dimethylsulfoxide, pyridine, chloroform,
dioxane, dichloromethane, tetrahydrofuran and suitable mixtures of
such solvents may be mentioned
The reaction temperature is selected from within the range known to
be emloyable for reactions leading to the formation of peptide
bonds, i.e. normally within the range of about -20.degree.C to
about 30.degree.C. Further, the precursor materials (protected
peptides) of the contemplated compounds according to this invention
may also be easily prepared by solid-phase synthetic processes.
After the contemplated condensation reaction has been completed, if
the product carries protective groups, they can be removed by
routine procedures. Among such routine procedures are catalytic
reduction in the presence of a catalyst such as palladium black,
palladium-on-carbon, platinum or the like, solvolysis by means of
hydrogen fluoride, trifluoroacetic acid or the like, and reduction
with metallic sodium in liquid ammonia.
The peptide (I) thus produced can be recovered from the reaction
product mixture by procedures known for the recovery of peptides,
e.g. by extraction, distribution, column chromatography, etc.
The present reaction may be carried out by per se conventional
solid phase method.
The peptide (I) may also be recovered in the form of a salt or
metal complex compound.
As acids which are able to form salts with peptide (I), there may
be mentioned inorganic acids such as hydrochloric acid, hydrobromic
acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid,
phosphoric acid, etc. and organic acids such as formic acid, acetic
acid, propionic acid, glycolic acid, lactic acid, pyruvic acid,
oxalic acid, malonic acid, succinic acid, maleic acid, fumaric
acid, anthranylic acid, cinnamic acid, naphthalenesulfonic acid or
sulfanylic acid, for instance.
The metals which are able to form metal complex compounds with
peptide (I) include, among others, zinc, nickel, cobalt, copper and
iron. Such a metal complex compound can be produced by conventional
procedures, for example, by reacting peptide (I) with the hydroxide
or oxide of a metal of the above-mentioned variety at pH about 6 to
8.
The polypeptides (I) according to this invention have the LH-RH
(luteinizing hormone releasing hormone) activity and, accordingly,
are able to promote the secretion of LH (luteinizing hormone) and
FSH (follicle stimulating hormone). Therefore, polypeptides (I) are
of use as drugs for promoting ovulations in women and other animals
(e.g. rats, ewes, pigs, cows, mares, quails or hens). The peptides
can also be used for other pharmaceutical purposes for which
conventional LH-RH, LH and FSH preparations have been employed.
Since the LH-RH activity of polypeptides (I) is about 10 to 25
times that of known naturally-occurring LH-RH, their dosage may be
determined for each application on the basis of the above multiple
whilst other factors (e.g. the subject of administration or the
kind of disease) are also taken into consideration. For example, a
suitable dosage may be selected from within the range of about 5
ng. (nano grams) to 10 .mu.g. daily per kilogram of body
weight.
Polypeptides (I) are primarily administered non-orally (e.g. by
injection or by the rectal or vaginal route), although they are
orally administered in certain instances.
The dosage forms employable include, for example, injections,
suppositories, pessaries and powders. The injections can be
prepared by dissolving about 10.gamma. to 500.gamma. of a
polypeptide (I) in 1 m. of physiological saline. Polypeptides (I)
can be also made into lyophilized ampoule products with mannitol
added as an excipient so that one may administer them as injections
for extemporaneous use.
The starting material peptides employable in the method of this
invention can be prepared either by known processes for peptide
synthesis or by utilizing such procecces as found necessary.
For further illustration of the invention, examples are given as
follows:
In the examples, the following abbreviations mean Rf value of a
thin layer chromatography on silica gel with the following solvent
system:
Rf.sup.1 : chloroform - methanol - acetic acid, 9:1:0.5
Rf.sup.2 : ethyl acetate - pyridine - acetic acid - water,
30:10:3:5
Rf.sup.3 : n-butanol - ethyl acetate - acetic acid - water,
1:1:1:1
EXAMPLE 1
Production of
(Pyr)Glu-His-Trp-Ser-Phe-(D)-Nva-Leu-Arg-Pro-Gly-NH.sub.2
a. Preparation of Z-(D)-Nva-Leu-Arg(NO.sub.2)-Pro-Gly-NH.sub.2
To a solution of Z-(D)-Nva-OH(380 mg),
H-Leu-Arg(NO.sub.2)-Pro-Gly-NH.sub.2 (690 mg.) and HONB (300 mg.)
in 5 ml. of dimethylformamide is added 340 mg. of DCC at 0.degree.C
with stirring. The mixture is stirred for 2 hours at 0.degree.C and
for additional 10 hours at room temperature. The reaction mixture
is filtered to remove the formed dicyclohexyl-urea, and the
filtrate is evaporated to dryness. The resulting residue is
dissolved in 100 ml. of chloroform and the solution is washed with
4 % aqueous sodium bicarbonate solution and water. The washed
solution is dried over anhydrous magnesium sulfate and evaporated
to dryness. The residue is then triturated with a mixture of
ethylacetate (25 ml.) and ether (25 ml.) to give a white powder,
which is collected by filtration and reprecipitated from
ethanol-ether: yield, 1.12 g., [.alpha.].sub.D.sup.23
-50.5.degree.(c=1.1 in Methanol), Rf.sup.1 =0.32.
Analysis for C.sub.32 H.sub.52 O.sub.9 N.sub.10 . calcd: C, 53.32;
H, 7.27; N, 19.43. Found: C, 53.49; H, 7.56; N, 19.19.
b. Preparation of
(Pyr)Glu-His-Trp-Ser-Phe-(D)-Nva-Leu-Arg-Pro-Gly-NH.sub.2
Z-(D)-Nva-Leu-Arg(NO.sub.2)-Pro-Gly-NH.sub.2 (1.0 g.) is dissolved
in 10 ml. of 25 % of hydrogenbromide in acetic acid, and the
solution is stirred for 50 minutes. The reaction mixture is diluted
with 200 ml. of dry ether to give a precipitate, which is collected
by filtration and washed well with dry ether. The collected powder
is dried over sodium hydroxide under reduced pressure. This powder
is dissolved in 10 ml. of dimethylformamide together with
(Pyr)Glu-His-Trp-Ser-Phe-OH(1.0 g.) and HONB (440 mg.). The
solution is cooled to 0.degree.C and to this is added DCC (400 mg.)
with stirring. The mixture is stirred for 6 hours at 0.degree.C and
for additional 16 hours at room temperature. The reaction mixture
is filtered to remove the formed dicyclohexyl-urea, and the
filtrate is triturated with addition of 100 ml. of ether to give a
precipitate, which is collected by filtration. The collected
precipitate is dissolved in 10 % aqueous ethanol and the solution
is applied on a column of polystyrene resin [Amberlite
XAD-2(150-250 mesh, 3.5 .times. 25 cm), Rohm & Haas Co. Ltd.
U.S.A.,] and the column is eluated by a gradient elution method
with 10 % aqueous ethanol and 100 % ethanol (500:550 ml.). The
principal fraction (380-520 ml.) is collected and evaporated to
dryness. The residue is dissolved in 5 ml. of hot methanol and
reprecipitated by addition of ethylacetate to give the protected
decapeptide amide. 1.41 g., [.alpha.].sub.D.sup.24 -35.4.degree.
(c=0.12, in methanol), Rf.sup.2 =0.20, Rf.sup.3 =0.56.
The protected decapeptide amide (500 mg.) is dissolved in 5 ml. of
anhydrous hydrogen fluoride together with 0.5 ml. of anisole at
-70.degree.C. After being stirred for 30 minutes at -5.degree.C,
hydrogen fluoride is removed by evaporation. The resulting residue
is dissolved in 20 ml. of water, and the solution is extracted
twice with 10 ml. of ethyl acetate. The aqueous layer is applied on
a column of carboxymethylcellulose (2 .times. 33 cm) and the column
is eluated by a gradient elution method using an ammonium acetate
buffer as eluate [0.005M, pH 6.8(500 ml.) - 0.2M, pH 6.8(500 ml.)].
The principal fraction (420 ml. - 680 ml.) is collected and
lyophilized to give a white powder. yield 380 mg.,
[.alpha.].sub.D.sup.22 -32.4.degree.(c=0.52, in 5 % acetic acid),
Rf.sup.2 =0.05, Rf.sup.3 =0.68.
Amino acid analysis: His 1.01; Arg 0.98; Trp 0.87; Ser 0.94; Glu
1.00; Pro 1.02; Gly 1.00; Nva 1.01; Leu 1.00; Phe 0.97 (peptide
content, 84 %).
EXAMPLE 2
Production of
(Pyr)Glu-His-Trp-Ser-Tyr-(D)-Nle-Leu-Arg-Pro-Gly-NH.sub.2 by
solid-phase procedure
a. Preparation of BOC-Gly-resin
In 60 ml. of chloroform-ethanol (2:1) is placed 10 g. of
chloromethyl-resin (Cl content 2.0 m mol/g.), followed by the
addition of 10.5 g. of BOC-Gly and 8.4 ml. of triethylamine. The
mixture is stirred at room temperature for 1.5 hours and, then,
heated for 24 hours. The resin is recovered by filtration and
washed well with dimethylformamide and further with ethanol, water,
ethanol and ether in the order mentioned and dried. Yield 17.55 g.
Amino acid analysis shows that this resin contains 0.88
millimols/gram of BOC-Gly.
b. Preparation of
(Pyr)Glu-His(Tos)-Trp-Ser(Bzl)-Tyr-(Bzl)-D-Nle-Leu-Arg(Tos)-Pro-Gly-resin
The reaction tank of an automatic peptide synthesizer (Model:
APS-800 of Simadzu Seisakusho K.K., Japan) is charged with 2.177 g.
of BOC-Gly-resin which is obtained in the above a) and caused to
swell with dichloromethane for 12 hours. Then, the following amino
acids are fed on the cycle given below.
BOC-Pro, BOC-Arg(Tos), BOC-Leu, BOC-D-Nle, BOC-Tyr(Bzl),
BOC-Ser(Bzl), BOC-Trp, BOC-His(Tos), (Pyr)Glu.
Dichloromethane (3 minutes .times. 3) .fwdarw. 50 % trifluoroacetic
acid/dichloromethane (10 min. and 30 min.) .fwdarw. dichloromethane
(3 min. .times. 3) .fwdarw. ethanol (3 min. .times. 3) .fwdarw.
dichloromethane (3 min. .times. 3) .fwdarw. 10 %
triethylamine/chloroform(10 min.) .fwdarw. chloroform (3 min.
.times. 3) .fwdarw. dichloromethane (3 min. .times. 2) .fwdarw.
BOC-amino acid-anhydride (synthesized from BOC-amino acid and DCC
by routine procedure) (30 min. and 60 min.) .fwdarw. acetylation
(with acetic anhydride in dichloromethane and triethylamine) (1
hour) .fwdarw. dichloromethane (3 min. .times. 3) [only (Pyr)Glu is
directly condensed with DCC in dimethylformamide].
Finally, the resin is washed with ethanol, chloroform and
dimethylformamide. Then, it is washed with ether and dried. Yield
6.20 g.
c. Preparation of
(Pyr)Glu-His-(Tos)-Trp-Ser(Bzl)-Tyr(Bzl)-D-Leu-Leu-Arg(Tos)-Pro-Gly-NH.sub
.2
In 50 ml. of ammonia-saturated methanol is suspended 2.622 g. of
the resin prepared in b) and the suspension is sitrred at room
temperature for 70 hours.
The resin is recovered by filtration and washed with
dimethylformamide. The filtrate and washings are pooled,
concentrated to dryness under reduced pressure and treated with
ether. The procedure gives 1.187 g. of crude powder.
A 588 mg. portion of this product is purified on a dry column of 50
g. of silica gel using as a developer a solvent mixture of methanol
and chloroform, whereupon 186 mg. of contemplated product is
obtained.
d. Preparation of
(Pyr)Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-Gly-NH.sub.2
In the presence of 0.2 ml. of anisole and 0.2 ml. of
mercaptoethanol, 173.3 mg. of the protected peptide obtained in c)
is dissolved in 5 ml. of dry hydrogen fluoride and the solution is
stirred at 0.degree.C for 1 hour. It is then concentrated to
dryness under reduced pressure and the concentrate is dissolved in
20 ml. of water. The insolubles are filtered off and the filtrate
is run down a column (1.5 cm dia. .times. 20 cm) of strongly basic
anion exchange resin (Amberlite IRA-410 acetate-form, Rohm &
Haas Co.Ltd.U.S.A.) Then, the effluent is purified by means of
carboxyl-methylcellulose (1.5 .times. 22 cm; the gradient elution
method using 0.005 M to 0.2 M ammonium acetate of pH 6.8) and
polystyrene resin (Amberlite XAD-2, Rohm & Haas Co.Ltd.U.S.A.)
(1.5 .times. 7.5 cm; the gradient elution method using 5 % to 70 %
aqueous ethanol). The eluate is further subjected to gelfiltration
chromatography on Sephadex LH-20 (Pharmacia Fine Chemicals, Sweden)
(0.9 .times. 53.5 cm; 0.1 N acetic acid). The procedure gives 39
mg. of contemplated compound. [.alpha.].sub.D.sup.24
-40.5.degree.(c=0.5 in 5 % aqueous acetic acid)
Amino acid analysis (acid hydrolysis in the presence of
thioglycolic acid): Glu 0.97; His 0.97; Trp 0.94; Ser 0.88; Tyr
1.0; Leu 1.00; Nle 1.06; Arg. 1.03; Pro 1.00; Gly 1.03 (87 %
recovery)
EXAMPLES 3-13
When similar procedures to those in Example 2 are followed except
for employing starting materials listed in the following table in
place of those in Example 2, the decapeptide amides (I) are
produced as listed in the table.
Table
__________________________________________________________________________
Decapeptide amide [.alpha.].sub.D.sup.24 (c=0.5 Starting materials
employed in Example (I) in 5% Amino acid analysis place of Example
2 R.sub.1 R.sub.2 R.sub.3 aqueous acetic acid BOC-Tyr-(Bzl)
BOC-D-Nle BOC-Leu
__________________________________________________________________________
His 1.02; Arg 1.01; 3 Tyr D-Ser Leu -44.8.degree. Trp 0.97; Ser
1.91; BOC-Tyr(Bzl) BOC-D-Ser BOC-Leu Glu 1.00; Pro 1.01; (Bzl) Gly
1.03; Leu 0.97; Tyr 0.96 His 0.96; Arg 1.00; Trp 0.89; Ser 0.92; 4
Tyr D-Abu Leu -43.2.degree. Glu 1.00; Pro 1.00; BOC-Tyr(Bzl)
BOC-D-Abu BOC-Leu Gly 0.99; Abu 0.97; Leu 1.00; Tyr 0.98 His 1.00;
Arg 0.99; Trp 0.92; Ser 0.91; 5 Tyr D-Nva Leu -38.9.degree. Glu
1.00; Pro 1.00; BOC-Tyr(Bzl) BOC-D-Nva BOC-Leu Gly 1.01; Nva 0.97;
Leu 0.98; Tyr 0.98 His 1.01; Arg 0.98; Trp 0.95; Thr 1.00; 6 Tyr
D-Thr Leu -37.5.degree. Ser 0.92; Glu 1.00; BOC-Tyr(Bzl) BOC-D-Thr
BOC-Leu Pro 1.00; Gly 0.99; Leu 1.00; Tyr 0.98 His 1.00; Arg 0.99;
Trp 0.87; Ser 0.89; 7 Tyr D-Phe Leu -52.4.degree. Glu 1.00; Pro
0.99; BOC-Tyr(Bzl) BOC-D-Phe BOC-Leu Gly 1.01; Leu 0.98; Phe 1.00;
Tyr 0.96 His 0.98; Arg 1.00; Trp 0.89; Ser 0.92; 8 Tyr D-Met Leu
-42.0.degree. Glu 0.99; Pro 0.98 BOC-Tyr(Bzl) BOC-D-Met BOC-Leu Gly
1.00; Met 0.79; Leu 1.00; Tyr 1.00 His 1.00; Arg 0.98; Trp 0.87;
Ser 0.98; 9 Phe D-Phe Leu -69.5.degree. Glu 1.01; Pro 0.98; BOC-Phe
BOC-D-Phe BOC-Leu Gly 1.00; Leu 1.00; Phe 1.98 His 1.00; Arg 1.00;
Trp 0.92; Ser 0.88; 10 Tyr D-Abu Ile -38.5.degree. Glu 1.03; Pro
1.00; BOC-Tyr(Bzl) BOC-D-Abu BOC-Ile Gly 1.00; Abu 0.96; Ile 0.98;
Tyr 0.98 His 0.96; Arg 0.94; Trp 0.87; Ser 1.87; 11 Phe D-Ser Nle
-32.5.degree. Glu 1.00; Pro 0.99; BOC-Phe BOC-D-Ser BOC-Nle Gly
1.00; Nle 0.96; (Bzl) Phe 1.02 His 1.00; Arg 1.01; Trp 0.81; Ser
0.89; 12 Phe D-Nle Nle -35.1.degree. Glu 1.02; Pro 1.00; BOC-Phe
BOC-D-Nle BOC-Nle Gly 1.00; Nle 2.03; Phe 0.97 His 0.96; Arg 1.00
Trp 0.86; Ser 0.89; 13 Phe D-Nva Ile -35.5.degree. Glu 1.00; Pro
1.00; BOC-Phe BOC-D-Nva BOC-Ile Gly 0.98; Nva 0.90; Ile 0.92; Phe
0.99
__________________________________________________________________________
* * * * *